The objectives of this proposal are to characterize and quantitate the functional consequences of s-CD4 domain structure. The thermodynamic solution stability of s-CD4, s-CD4 domain fragments and mutants of these proteins will be studied; the modulation of gp120 recognition by domain interactions within s-CD4 will be quantitated; the structural basis for the relationship between s-CD4 domain structure and gp120 recognition will be determined; assays will be developed to screen and identify peptides or other small molecules that disrupt the CD4-gp120 interaction. Domain communication can influence protein stability, ligand binding, and signal transduction. The structural information on CD4 obtained from the proposed studies will be useful for the rational development of potential new drugs to treat AIDS and will be important for understanding the function of CD4 as a membrane receptor. The AIDS virus infection is initiated by the binding of the HIV envelope protein, gp120, to the T cell membrane-bound antigen, CD4. Based on primary and secondary structural homologies, the structure of the extracellular portion of CD4 is thought to be a tandem repeat of four immunoglobulin-like domains, V1-V4. Recent crystal structures of V1V2 support this hypothesis. The binding site for gp120 has been identified to be within V1, but the crystal structure of V1V2 reveals an intimate association, between the domains, which is likely to modulate domain stability and ligand binding to either domain (also suggested from gp120 and antibody binding studies). Several kinds of related information are sought in the studies proposed. (1) The structural stability of soluble-(s-) CD4 and domain fragments V1, V2 and V1V2 and the domain structure of s-CD4 will be studied by differential scanning calorimetry. (2) The oligomerization state of all macromolecules under study will be determined by analytical ultracentrifugation. (3) The domain interaction free energy between V1 and V2 will be determined using titration calorimetry and analytical affinity chromatography. (4) Interaction of s-CD4, V1, V2 and V1V2 with gp120 will be quantitated using titration calorimetry, analytical affinity chromatography and other affinity methods. The effect of domain interactions within s-CD4 on gp120 recognition, as well as the effects of peptides or other small molecules on s-CD4-gp120 interactions will come from these studies. In addition to native-sequence s-CD4 and domain fragments, mutants of s-CD4 and domain fragments will be studied to better correlate structure with protein stability. Also, we have begun to use the V1V2 crystal structure to design new mutants to establish the quantitative importance of specific residues in V1, including residues at the V1-V2 interface, to gp120 recognition.